Polymerized whey protein encapsulated antioxidant compound and a process for preparation of same

ABSTRACT

A process is provided for encapsulating glutathione (GSH), 3,3′-diindolylmethane (DIM), coenzyme-Q10 (CoQ10), and other hydrophobic antioxidant compounds by using whey proteins which may be polymerized in a particular manner. Further, compositions comprising polymerized whey protein (PWP) encapsulated glutathione, polymerized whey protein (PWP) encapsulated CoQ10, and polymerized whey protein (PWP) encapsulated DIM are provided.

This application claims the benefit of U.S. Provisional application No.63/067,091, filed on Aug. 18, 2020, which is hereby incorporated byreference herein.

TECHNICAL FIELD

The present invention relates to a process for encapsulating glutathione(GSH), 3,3′-diindolylmethane (DIM), CoEnzyme Q10 (CoQ10), and otherhydrophobic antioxidant compounds by using whey proteins which may bepolymerized in a particular manner. Herein are described compositionscomprising polymerized whey protein encapsulated glutathione, DIM,CoEnzyme Q10, and the like.

BACKGROUND

Glutathione (GSH) is an antioxidant found in plants, animals, fungi, andsome bacteria. Glutathione is the most abundant thiol in animal cells,ranging from 0.5 to 10 mM. It is present both in the cytosol and theorganelles (Guoyao Wu, Yun-Zhong Fang, Sheng Yang, Joanne R. Lupton,Nancy D. Turner. “Glutathione Metabolism and its Implications forHealth,” Journal of Nutrition (2004) 134 (3): 489-92).

Glutathione exists in reduced (GSH) and oxidized (GSSG) states. Theratio of reduced glutathione to oxidized glutathione within cells is ameasure of cellular oxidative stress where increased GSSG to GSH ratiois indicative of greater oxidative stress (A. Pastore, et al.,“Determination of blood total, reduced, and oxidized glutathione inpediatric subjects”. Clinical Chemistry (August 2001) 47 (8): 1467-9; SCLu. “Glutathione synthesis”. Biochimica et Biophysica Acta (BBA)—GeneralSubjects (May 2013) 1830 (5): 3143-53). In healthy cells and tissues,more than 90% of the total glutathione pool is in the reduced form(GSH), with the remainder in the disulfide form (GSSG); (K. M. Halprin,A. Ohkawara “The measurement of glutathione in human epidermis usingglutathione reductase”. The Journal of Investigative Dermatology (1967)48 (2): 149-52).

GSH protects cells by neutralizing (i.e., reducing) reactive oxygenspecies. This conversion is illustrated by the reduction of peroxides:2GSH+R2O2→GSSG+2ROH(R=H,alkyl)

Also, free radicals may be quenched in vivo:GSH+R·→0.5GSSG+RH

In addition, glutathione plays a key role in cellular regulation andmetabolism. With respect to these key metabolic and cellular roles, itwould be advantageous to provide glutathione in a stable andbioavailable composition. Glutathione suffers from a lack of oralbioavailability in the digestive tract. Previous encapsulationtechnologies have been developed, predominately using lipidencapsulation (such as lecithin) in conjunction with syntheticchemicals, such as polysorbate 80, in order to increase absorption ofdifficult to absorb compounds, but the application of whey protein orpolymerized whey protein has not, until now, been applied in this way.Therefore, a safe, synthetic chemical free option is needed to increasebioavailability.

If a way could be found to improve stability and decrease degradation ofglutathione in a composition for delivery to a mammal, in particular ahuman subject, this would provide a useful contribution to the art.Further, if a way could be found to optimize absorption of glutathioneand utilization by the body, e.g. increasing bioavailability, this wouldprovide a further useful contribution to the art

Regarding encapsulation of nutrients, certain film-forming compounds andsurfactants are useful, for example, polyvinylpyrrolidone,polyoxyethylene stearate, sodium cholate, deoxycholate and taurocholatephosphatidyl choline, dioleoyl phosphatidyl choline,phosphatidylglycerol, dioleoylphosphatidylglycerol,dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine,phosphatidylethanolamine, phosphatidylserine, sphingomyelin,methylcellulose, hydroxypropyl methylcellulose, hydroxyethylcellulose,hvdroxypropylethylcellulose, one or more of which may be blended withlecithin.

3,3′-diindolylmethane (“DIM”) is an active metabolite ofindole-3-carbinol derived from cruciferous vegetables and exhibits abroad spectrum of anticancer properties. The stability of DIM is a majorchallenge in the pharmaceutical industry. Moreover, DIM has poor oralbioavailability due to its low solubility and high lipophilicity.Encapsulation by whey protein is known in order to develop nanoparticleswith controlled size and properties, which process may provide for theprotection, preservation, and delivery of sensitive compounds such asaroma or nutraceuticals.

Certain methods are known for encapsulating 3,3′-diindolylmethane(“DIM”) and like species using ultrasound. See, A. Khan, et al.,“Physicochemical and Microstructural Properties of Polymerized WheyProtein Encapsulated 3,3′-Diindolylmethane Nanoparticles,” Molecules(2019) 24:702.

If a way could be found to improve the encapsulation process to provide,for example, a polymerized whey protein encapsulated DIM having betterstability, solubility and other improved properties such as oralbioavailability, this would provide a contribution to the chemical andformulation arts.

SUMMARY OF THE INVENTION

A composition is described including polymerized whey proteinencapsulating glutathione.

A process is described for making polymerized whey proteinmicroencapsulated glutathione, comprising the steps of: dissolving wheyprotein concentrate powder in water at about 8-12% w/v to provide anaqueous solution; heating the whey protein concentrate solution to atleast 80° C. for about 15-25 minutes to provide a polymerized wheyprotein solution; adding an antioxidant compound to the polymerized wheyprotein solution during the cooling process; stirring with a homogenizeror shear mixer to provide a clear homogeneous solution; and isolatingpolymerized whey protein microencapsulated antioxidant compound, forexample, by freeze-drying or spray-drying to provide a powder.

Further, a process is described for making polymerized whey proteinmicroencapsulated DIM (PWP-DIM), comprising the steps of: (a) dissolvingwhey protein concentrate powder in water at 10% w/v to provide anaqueous solution; (b) heating the whey protein concentrate solution toat least about 70-80° C. to provide a polymerized whey protein solution;(c) adding DIM to the polymerized whey protein solution; (d) adjustingthe pH in a range from about 6.5 to about 9.0; (e) cooling thepolymerized whey protein solution; (f) stirring during cooling fromabout 80° C. to about 45° C. to provide a clear homogeneous solution;and (g) isolating polymerized whey protein microencapsulated DIM.

One objective is to prepare a PWP-DIM having a visibly better encasementfor greater protection through the digestive tract, i.e. the GI tract.

Another objective is to prepare a PWP-DIM having improved absorption inthe digestive tract.

Further, a process is described for making polymerized whey proteinmicroencapsulated antioxidant compound, comprising the steps of: (a)dissolving whey protein concentrate powder in water at 10% w/v toprovide an aqueous solution; (b) heating the whey protein concentratesolution to about 70-80° C. for about 15 minutes to provide apolymerized whey protein solution; (c) adding glutathione to thepolymerized whey protein solution; (d) stirring to provide a clearhomogeneous solution; and (e) isolating polymerized whey proteinmicroencapsulated glutathione, for example, by freeze-drying to providea powder.

In one embodiment, the process for making the polymerized whey proteinmicroencapsulated antioxidant compound is used to make polymerized wheyprotein microencapsulated glutathione (PWP-GSH).

In another embodiment, the process for making the polymerized wheyprotein microencapsulated antioxidant compound is used to makepolymerized whey protein microencapsulated coenzyme-Q10 (PWP-CoQ10).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a standard curve for determining reduce glutathione (GSH)using HPLC. The standard curve was plotted as a function ofconcentrations to peak area.

FIG. 2 depicts a standard curve for determining reduce glutathione (GSH)using an assay kit. The standard solutions were plotted as absolute ODvalue vs GSH concentration (μM).

FIG. 3 depicts reduced glutathione (GSH) plasma concentration (mon) as afunction of time after oral administration of test samples in mice.

FIG. 4 depicts a standard curve for determining the antioxidant activityof plasma. The standard curve was plotted by determining the OD valuesof different standard samples at 0.1, 0.2, 0.4, 0.8, and 1.0 mM.

FIG. 5 depicts, in an embodiment, the in vivo antioxidant activity ofplasma of mice after oral administration of free GSH, WPC/PWPC basedGSH, WPC/PWPC, as measured by assay kit (ABTS method). The order of thevertical bars at individual time points, from left to right: Control,WPC, PWP, GSH, WPC-GSH, and PWP-GSH.

FIGS. 6A-6F depicts, in another embodiment, GSH concentration (μM) inthe tissues of mice measured after oral administration of free GSH,WPC/PWPC based GSH, WPC/PWPC. The order of the vertical bars atindividual time points, from left to right: Control, WPC, PWP, GSH,WPC-GSH, and PWP-GSH. Figures A-F show measurements in brain (A), heart(B), lung (C), kidney (D), liver (E), and intestine (F).

FIG. 7 depicts, in another embodiment, body weight curves in rats ingrams in 28-day feeding test incorporating PWP-GSH in feed.

FIG. 8 depicts, in another embodiment, food consumption curves in ratsin grams/rat/day in 28-day feeding test incorporating PWP-GSH in feed.

FIG. 9 depicts, in another embodiment, food efficiency curves in rats in28-day feeding test incorporating PWP-GSH in feed.

FIG. 10A depicts, in an embodiment, microscope images of stainedsections of several organ tissues in male rats in 28-day feeding testincorporating 4% by wt. PWP-GSH in feed vs. control.

FIG. 10B depicts, in an embodiment, microscope images of stainedsections of several organ tissues in female rats in 28-day feeding testincorporating 4% by wt. PWP-GSH in feed vs. control.

FIG. 11 depicts a standard curve for determining reduced GSH content ina solid sample using a reduced glutathione (GSH) assay kit (A006-2-1).

FIG. 12 depicts TEM images of PWPC and PWPC-GSH determined usingstandard techniques. Gradations at bottom are 1.0 micrometer.

FIG. 13A depicts particle size distribution of whey protein encapsulatedglutathione nanoparticles, for example, PWPC-GSH, based on WPC.

FIG. 13B depicts particle size distribution of whey protein encapsulatedglutathione nanoparticles, for example, PWPI-GSH, based on WPI.

FIG. 14 depicts polydispersity index (PDI) of whey protein encapsulatedglutathione nanoparticles, for example, PWPI-GSH, based on WPC and WPIstarting materials. Letters a-c meaning significant (P≤0.05).

FIG. 15 depicts Zeta potential (mV) of whey protein encapsulatedglutathione nanoparticles, for example, PWPI-GSH, based on WPC and WPIstarting materials. Letters a-d meaning significant (P≤0.05).

FIG. 16A depicts Apparent viscosity (mPa-sec) vs. Shear rate (1/s) ofwhey protein encapsulated glutathione nanoparticles, for example,PWPC-GSH, based on WPC.

FIG. 16B depicts Apparent viscosity (mPa-sec) vs. Shear rate (1/s) ofwhey protein encapsulated glutathione nanoparticles, for example,PWPI-GSH, based on WPI.

FIG. 17A depicts Circular dichroism [Θ]×10³ deg cm² dmol⁻¹ vs.wavelength (nm) of whey protein encapsulated glutathione nanoparticles,for example, PWPC-GSH, based on WPC.

FIG. 17B depicts Circular dichroism [Θ]×10³ deg cm² dmol⁻¹ vs.wavelength (nm) of whey protein encapsulated glutathione nanoparticles,for example, PWPI-GSH, based on WPI.

FIG. 18A depicts FT-IR spectra of whey protein encapsulated glutathionenanoparticles, for example, PWPC-GSH, based on WPC, obtained usingstandard techniques.

FIG. 18B depicts FT-IR spectra of whey protein encapsulated glutathionenanoparticles, for example, PWPI-GSH, based on WPI, obtained usingstandard techniques.

FIG. 19 depicts average particle size (nm) measured by the standardmethod for PWPI std., PWPI-CoQ10 samples in ratios of 100:1, 80:1, 60:1,40:1, and 20:1 (PWPI:CoQ10).

FIG. 20 depicts polydispersity index (PDI) measured by the standardmethod for PWPI std., PWPI-CoQ10 samples in ratios of 100:1, 80:1, 60:1,40:1, and 20:1 (PWPI:CoQ10).

FIG. 21 depicts Zeta potential (mV) measured by the standard method forPWPI std., PWPI-CoQ10 samples in ratios of 100:1, 80:1, 60:1, 40:1, and20:1 (PWPI:CoQ10).

DETAILED DESCRIPTION

In one aspect, the invention relates to microencapsulation of both fatand water soluble antioxidants with polymerized whey protein for theexpressed purpose of protecting the nutrients from degradation andoptimizing absorption and utilization in the body. For example, thepresent inventors, have contributed to and described the present methodfor preparation of polymerized whey protein encapsulated glutathione(PWP-GSH) in S. Zhang, et al., “Polymerized Whey ProteinConcentrate-Based Glutathione Delivery System: PhysicochemicalCharacterization, Bioavailability and Sub-Chronic Toxicity Evaluation,”Molecules (2021) 26:1824, hereby incorporated by reference in itsentirety.

In a further aspect, the invention relates to a novel process ofmanipulating whey protein, by adjusting temperature and pH, to allow forencapsulation of the protein around nutrients, specificallyantioxidants, for protection and optimal absorption and utilization. Theantioxidants that have been successfully encapsulated using the presentmethod are glutathione, diindolylmethane, and coenzyme Q10. Currentstudies are underway to apply this technology for use in dietarysupplements (e.g., to be tableted, encapsulated, incorporated into aready to drink powder) and to allow antioxidants to be successfullyincorporated into a variety of food and beverages to increase the foodor beverage's health benefit.

The advantages of the current process is that a technically simplifiedand more natural process, without the use of certain chemical agentswhich are typically not desired by natural food or dietary supplementcompanies, has been realized.

In a principal embodiment, the encasement of the antioxidant compounds,and other derivatives, is more evenly uniform which allows for betterprotection of the active component in the digestive tract. Further, thisprocess embodiment provides better encasement when viewedmicroscopically, for example using SEM or TEM.

The PWP-GSH compositions and formulations described herein demonstratedimproved absorption in vivo, better pharmacokinetics, betterbioavailability, and higher antioxidant capacity when compared to freeGSH. The PWP-GSH compositions and formulations described herein alsodemonstrated improved absorption in vivo, better pharmacokinetics, andbetter bioavailability when compared to comparator GSH product.

Microencapsulation

Microencapsulation is a technique used for the protection of a widerange of biomolecules. See, Whey Protein Production, Chemistry,Functionality, and Applications, Ed. Mingruo Guo (one of the presentinventors) Chap. 7, “Whey Protein Functional Properties and Applicationsin Food Formulation,” pp. 157-204 (and references cited therein),(Wiley: Hoboken, N.J., 2019), which is incorporated by reference herein.

Formulations may be prepared as any product form suitable for use inhuman individuals, including reconstitutable powders, ready-to-feedliquids, parenteral (intravenous) formulations, and dilutable liquidconcentrates, product forms which are all well known in the nutritionalformula art. As used in the present application, the amounts ofcomponents present in formulations or compositions refer to the amountswhen the formulation or composition is ready for consumption by thehuman individual.

Formulations or compositions can optionally be sterilized andsubsequently used on a ready-to-feed basis, or can be stored asconcentrates. Concentrates can be prepared by spray drying a liquidformulation prepared as above, and a formulation can be reconstituted byrehydrating the concentrate. The formulation concentrate is a stableliquid and has a suitable shelf life.

For powder embodiments of formulations or compositions comprising GSH,whey protein encapsulated GSH (WP-GSH), or polymerized whey proteinencapsulated GSH (PWP-GSH), used in the methods of the presentinvention, reconstitution of the powder can be done with a suitableaqueous liquid, preferably water. Reconstitutable powders are typicallyin the form of flowable or substantially flowable particulatecompositions, or at least particular compositions that can be easilyscooped and measured with a spoon or similar other device, wherein thecompositions can be easily reconstituted by the intended user with asuitable aqueous fluid, typically water, to form a liquid formulation orcomposition. In this context, “immediate” use generally means withinabout 48 hours, most typically within about 24 hours, preferably rightafter reconstitution. These powder embodiments include spray dried,agglomerated, dry mixed or other known or otherwise effectiveparticulate form. The quantity of a nutritional powder required toproduce a volume suitable for one serving can vary.

The nutritional formulas used in the methods of the present inventionmay be packaged and sealed in single or multi-use containers, and thenstored under ambient conditions for up to about 36 months or longer,more typically from about 12 to about 24 months. For multi-usecontainers, these packages can be opened and then covered for repeateduse by the ultimate user, provided that the covered package is thenstored under ambient conditions (e.g., avoid extreme temperatures) andthe contents used within about one month or so.

Compositions for oral formulations useful for delivering a dietarysupplement composition comprising GSH, whey protein encapsulated GSH(WP-GSH), or polymerized whey protein encapsulated GSH (PWP-GSH), can beorally administered, for example, with an inert diluent or with anassimilable edible carrier, or it can be enclosed in hard or soft shellgelatin or hydroxypropyl methylcellose (i.e., hypromellose) capsules, orit can be compressed into tablets, or it can be incorporated directlywith the food of the diet. For oral administration, a dietarycomposition comprising GSH, whey protein encapsulated GSH (WP-GSH), orpolymerized whey protein encapsulated GSH (PWP-GSH), may be incorporatedwith an excipient and used in the form of ingestible tablets, buccaltablets, troches, capsules, elixirs, suspensions, syrups, wafers, andthe like. The tablets, troches, pills, capsules, and the like can alsocontain the following: a binder such as gum tragacanth, acacia, cornstarch, or gelatin; excipients such as dicalcium phosphate,microcrystalline cellulose, and the like; a disintegrating agent such aspotato starch, alginic acid, and the like; a lubricant such as magnesiumstearate; and a sweetening agent such as sucrose, lactose, or saccharincan be added or a flavoring agent such as peppermint, oil ofwintergreen, or cherry flavoring. When the dosage unit form is acapsule, it can contain, in addition to materials of the above type, aliquid carrier. Various other materials can be present as coatings or tootherwise modify the physical form of the dosage unit. For instance,tablets, pills, or capsules can be coated with shellac, sugar, or both.A syrup or elixir can contain the active compound, sucrose as asweetening agent, methyl and propylparabens as preservatives, a dye, andflavoring such as cherry or orange flavor. Oil-in-water emulsions may bebetter for oral use in infants because these are water-miscible, andthus their oiliness is masked. Such emulsions are well known in thepharmaceutical sciences.

Determination of GSH using HPLC

Optimum conditions for HPLC analysis:

Column: symmetry C18

Mobile phase: Water:Acetonitrile=90:10

Flow rate: 0.5 ml/min

Wavelength: 218 nm (UV detector)

Injection volume: 0.5 μL

GSH powder was dissolved in ultra-pure water to make a stock solution of10 mg/ml. A series of standard solutions (0.2 mg/ml, 0.4 mg/ml, 0.6mg/ml, 0.8 mg/ml, 1.0 mg/ml) were obtained by dilution of the stocksolutions using ultra-pure water. GSH was determined using HPLC. Thestandard curve was plotted as a function of concentrations to peak areaas shown in FIG. 1 .

The compositions and methods described in the embodiments above may befurther understood in connection with the following Examples. Inaddition, the following non-limiting examples are provided to illustratethe invention. However, the person skilled in the art will appreciatethat it may be necessary to vary the procedures for any given embodimentof the invention, e.g., vary the order or steps of the methods.

Example 1

Whey Protein-GSH Mixture (WP-GSH)

Whey protein concentrate (WPC) solutions (10% protein, w/v) wereprepared by dissolving whey protein concentrate powder in deionizedwater at room temperature and then stirred (700 rpm) for 2 h. The stocksolution was stored at 4° C. overnight for complete hydration. Wheyprotein solution was warmed up to ambient temperature and then mixedwith reduced-GSH at weight ratios of WPC (powder): GSH=1:1, 1:1.5 and1:2. The mixtures were stirred for 20 min to achieve completedissolution.

Polymerized Whey Protein Encapsulated GSH (PWP-GSH)

Whey protein concentrate (WPC) solutions (10% protein, w/v) wereprepared by dissolving whey protein concentrate powder in deionizedwater at room temperature and then stirred (700 rpm) for 2 h. Thesolution was stored at 4° C. overnight for complete hydration. Wheyprotein solutions were returned to ambient temperature and the pH wasadjusted to 7 or 8 and then heated at 80° C. for 15 min. Polymerizedwhey protein (PWPC) solutions were obtained by cooling the heated wheyprotein solutions in mixed water-ice quickly to room temperature (25±1°C.). The PWPC solutions were then mixed with GSH powder at weight ratiosof PWPC:GSH=1:1, 1:1.5 and 1:2. The mixtures were mixed for 20 min toachieve complete dissolution.

Stability Experiments

All whey protein based GSH solutions were observed for stability for 20h at room temperature. Table 1 below lists the stability of whey proteinbased glutathione solutions, including polymerized embodiments.

TABLE 1 WPC/ PWPC:GSH Time Sample (w/w) 0 h 4 h 16 h 20 h 12% 1:1   NoNo No No WPC sediment sediment sediment sediment, viscous 1:1.5 Almostno Little More More sediment sediment sediment sediment 1:2   More MoreSediment Sediment sediment sediment increase increase 10% 1:1   No No NoNo WPC sediment sediment sediment, sediment, running running well well1:1.5 No No No No sediment sediment sediment sediment 1:2   More MoreSediment Sediment sediment sediment increase increase 10% 1:1   No NoGel Gel PWPC sediment sediment (pH7) 1:1.5 No No Gel Gel sedimentsediment 1:2   More More Gel Gel sediment sediment 10% 1:1   No No GelGel PWPC sediment sediment (pH8) 1:1.5 No No Gel Gel sediment sediment1:2   More More Gel Gel sediment sediment WPC (pH 7 and pH 8) at theconcentration of 12% gelled after heating.

As shown in Table 1, the PWP-GSH samples are stable to at least 4 hours.

Example 2

Pharmacokinetic Study

ICR mice, male, SPF, 3 weeks, weighing from 18 to 22 g were provided byBeijing HFK Bioscience Co., Ltd (Beijing, China). Reduced glutathione(GSH) assay kit (A006-2-1), total antioxidative capacity measurement kit(ABTS method) (A015-2-1) were purchased from Nanjing Jianchengbioengineering institute (Nanjing, Jiangsu China).

All mice were housed in plastic lab animal cages in a ventilated room.The room was maintained at 20±2° C. and 60±10% relative humidity with a12 h light/dark cycle. Water and commercial laboratory complete food formice were available ad libitum. They were acclimated to this environmentfor 7 days before treatment. All animal experiments were approved by theAnimal Welfare and Research Ethics Committee at Jilin University(Approval ID: SY201905018).

Blood collection. Before blood collection, 30 μL heparin solution wasadded to 1.5 mL centrifugal tubes and vortexed. The blood samples (about0.5 mL) was collected by removing one eye of the mice. The blood wascentrifuged at 6000 rpm at room temperature for 2 min. The upper plasmawas transferred to a new centrifugal tube. The plasma concentration ofGSH was analyzed using the GSH assay kit.

GSH stock solution (1 mmol/L) was diluted to a series of standardsolutions at concentrations of 0 μmol/L, 5 μmol/L, 10 μmol/L, 20 μmol/Land 100 μmol/L. The standard solutions were plotted as absolute OD valuevs GSH concentration (FIG. 2 ).

A. The blood samples were collected after oral administration of freeGSH, whey protein based GSH, whey protein, polymerized whey proteinbased GSH and polymerized whey protein by gavage at several time points(0, 15 min, 30 min, 1 h, 2 h and 4 h) in each group of 6 mice. The doseof the whey protein based GSH is adjusted to have an equivalent amountof 100 mg/kg of GSH. The GSH concentration in the blood samples aredetermined by assay kit.

Table 2 shows test groups and gavage volume.

TABLE 2 10% 10% 10% 10% Sample Control WPC PWPC WPC-GSH PWPC-GSH GSHGavage 0.3 mL 0.3 mL 0.3 mL 0.3 mL 0.3 mL 0.3 mL Volume saline

As shown in FIG. 3 , PWP-GSH test material provides an excellent andsignificant increase in concentration of GSH in plasma after oraladministration.

B. Antioxidant Activity In Vivo

Trolox stock solution (10 mM) was diluted to 0.1, 0.2, 0.4, 0.8, 1.0 mM.The standard curve was plotted by determining the OD values of differentstandard samples (FIG. 4 ). The antioxidant activity of plasma wasexpressed as the fold of capacity to Trolox with the TAOC (totalantioxidative capacity) of Trolox as 1.

The total antioxidative capacity of all samples were also measured usingthe assay kit and the results are shown in FIG. 5 . In vivo antioxidantactivity of plasma of mice after oral administration of free GSH,WPC/PWPC based GSH, WPC/PWPC, was measured by assay kit (ABTS method).

As shown in FIG. 5 , PWP-GSH sample over time exhibits the most robustantioxidant activity.

C. Tissue Distribution of GSH

To evaluate delivery efficiency of GSH to the organs, the tissue samplesof brain, heart, kidney, liver, lung, and intestine were collected at 0,1, 2, 4, and 6 h post-oral administration in each group of 6 mice. Thetissues were homogenized, protein precipitated, centrifuged, and thenanalyzed for GSH content using the assay kit (FIGS. 6A-6F).

As shown in FIGS. 6A-6F, distribution of free GSH in various tissues wassignificantly greater than control. Further, distribution of GSH for thePWP-GSH group in various tissues was significantly greater than control.

D. Toxicity Study of Polymerized Whey Protein Based GSH (PWP-GSH)

Whey protein concentrates were provided by Fonterra Co-operative Group(Auckland, New Zealand). Pentobarbital sodium, formalin and absoluteethanol were provided by Beijing Works (Beijing, China). SD rats at week3 were provided by Beijing HFK Bioscience Co., Ltd (Beijing, China).

Diet Formulations

Polymerized whey protein concentrate based GSH (PWP-GSH) was preparedaccording to Example 1 with polymerized whey protein concentration of10% and whey protein and GSH ratio of 1:1. The prepared polymerized wheyprotein GSH was then dried using a freeze drier (Alpha 1-4 LDplus,Germany). Then the powdered polymerized whey protein based GSH wasincorporated into normal feeds at the percentage of 0.5%, 1% and 4%(w/w) which corresponds to 0.25%, 1% and 2% percentage for GSH. Thediets were prepared by Beijing HFK Bioscience Co., Ltd (Beijing, China).The dose was set based on the daily intake for health human (100 mg perday). The dose for human will be 1.6 mg/kg calculated with averageweight of 60 Kg. According to the conversion equation, it equals to 10mg/kg for rats. The does was set to be 0.5%, 1% and 4% which arecorresponding to 25, 50 and 200 folds of human daily intake.

Experimental Design

Eighty rats (male and female for half) at age of 3 weeks were purchasedfrom Beijing HFK Bioscience Co., Ltd (Beijing, China). All rats werehoused in plastic lab animal cages in a ventilated room. The room wasmaintained at 20±2° C. and 60±10% relative humidity with a 12 hlight/dark cycle. Water was available ad libitum. Subject rats wereacclimated to this environment for 7 days before treatment in whichthere were no apparent changes in general status. Followingacclimatization, rats were randomly allocated to four groups (10 per sexper group) based on body weight means. Individual body weight of a groupat randomization was within ±20% of the overall mean. Compared to theanimals in control groups, the low, mid and high-dose group animalsreceived 0.5%, 1% and 4% whey protein based GSH in their diets,respectively. All animal experiments were approved by the Animal Welfareand Research Ethics Committee at Jilin University (Approval ID:SY201905018).

Clinical Observations

Coat condition, skin, mucous membranes, occurrence of secretions andexcretions, autonomic nervous system activity, changes in gait, andposture of each rat were observed throughout the study.

Body Weight and Food Intake

Individual body weights as well as body weights at the interval of 4days were weighed and recorded for a total period of 28 days. Final bodyweights (fasted) were recorded prior to the scheduled necropsy. Feedintake was also measured and expressed as the mean food consumption(expressed as g/rat/day) was calculated for the corresponding intervals.

Blood Collection

At the termination of the experiment, all animals were fasted for 12 hprior to blood collection but did have access to water. Rats wereinjected for 2% pentobarbital sodium solution at the level of 0.2 ml/100g. Then, two separate blood samples for hematology and serum chemistrywere collected via heart. For hematology analysis, the blood sampleswere collected by EDTA-2K coated tubes and then determined for whiteblood cells (WBC, red blood cells (RBC), hemoglobin (HGB), hematocrit(HCT), blood platelet count (PLT), mean corpuscular volume (MCV), meancorpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration(MCHC), red cell volume distribution (RDW), mean platelet volume (MPV)lymphocyte (LYM), neutrophilicgranulocyte (GRAN), monocyte (MONO),Lymphocyte (LYM %), granulocyte (GRA), Percent monocytes (MON %) usingExigo animal hematology analyzer.

For serum chemistry analysis, the blood sample was centrifuged at 10000rpm at room temperature for 3 min. The upper serum was transferred to anew centrifugal tube and then determined for albumin (ALB), calcium(Ca), creatinine (Crea), total bilirubin (TB), TotalProtein (TP),inorganic phosphorus (PHOS), urea (UREA), amylase (AMY), triglyceride(TG), glucose (GLU), the ratio of BUN to CR (U/C), creatine kinase (CK),Globulin (GLOB), aspartate aminotransferase (AST) using Smt-120vautomatic biochemical analyzer.

Organ Weights, Gross Necropsy and Histopathology

At termination, all the rats were anaesthetized by pentobarbital sodiumand exsanguinated by transecting the anocelia. Then a complete grosspathology examination was conducted by visual inspection duringnecropsy. Brain, heart, lung, liver, spleen, kidney, bladder, ovary,uterus, testes, epididymis and seminal vesicles for all animals wereexcised and weighed. Relative weight of each organ (or paired organs)was calculated based on final individual body weight measured on the dayof termination. Tissue sections from these organs were fixed with 10%buffered formaldehyde except testes were fixed in Bouin solution,embedded in paraffin, sectioned at 2-5 μm, mounted on glass microscopeslides, stained with standard hematoxylin-eosin and examined using lightmicroscopy. All the histopathology procedures were carried out inCollege of Animal Science and Veterinary Medicine, Jilin University.

Results

During the course of the experiment, there was no observed adverseeffects in the experimental group compared with the control group.

During the course of the experiment, no treatment-related signs ofadverse effects in clinical appearance of the animals were observed.Body weight increased gradually as the treatment period progressed (FIG.7 ). There was no statistically significant difference in body weightbetween female groups. For male groups, from 16 days, 1% male groupswere significantly different from those of control. The body weightchanges were observed only in male group and there was no dose-dependenteffect.

Results for food consumption and food efficiency of rats for 28 days areshown in FIGS. 8 and 9 . There was no PWPC-GSH related toxicity effectobserved in experimental groups although there was some significantdifference between experimental group and control at some time point.Groups of 0.5% and 4% female showed significant difference in foodefficiency compared with control at 8^(th) day (p<0.05).

Table 3 shows serum biochemistry for male rats in the 28-day toxicitystudy.

TABLE 3 0% 0.5% 1% 4% Albumin (g/L) 34.78 ± 1.05 33.90 ± 1.34 33.53 ±0.99 33.01 ± 1.34* Total protein (g/L) 66.39 ± 4.69 63.52 ± 3.90 62.75 ±2.46 59.11 ± 7.98  Globulin (g/L) 32.71 ± 3.08 29.62 ± 3.13 29.22 ± 2.36 28.1 ± 2.77* Globulin ratio  1.12 ± 0.14  1.15 ± 0.11  1.16 ± 0.10 1.18± 0.10 Total bilirubin (μmol/L) <1.0 <1.0 <1.0 <1.0 Aspartate 138.20 ±17.63  105.00 ± 11.51*  81.60 ± 10.14* 91.17 ± 8.13* aminotransferase(U/L) Alanine 45.70 ± 5.85 43.89 ± 6.31 40.50 ± 4.55   39 ± 7.73*aminotransferase (U/L) Amylase (U/L) 2320.37 ± 178.97  1898.71 ± 145.35* 2009.2 ± 194.24*  1775.0 ± 110.55* Creatinine (μmol/L) <4 <4 < 4 <4Creatine kinase (U/L) 1110.00 ± 92.28   723.00 ± 64.50*  354.00 ±32.04**  413.40 ± 31.05** Triglyceride (μmol/L)  0.77 ± 0.37  0.51 ±0.26  0.56 ± 0.17 <0.3 Glucose (μmol/L)  7.92 ± 0.95  7.17 ± 0.51  7.03± 1.12   6.203 ± 0.51833* Calcium (μmol/L)  2.57 ± 0.05  2.50 ± 0.08  2.45 ± 0.0 **  2.379 ± 0.10** Inorganic phosphorus  3.35 ± 0.31  3.03± 0.36  3.25 ± 0.48 2.70 ± 0.23 (μmol/L) BUN (μmol/L)  5.06 ± 0.58  4.52± 0.69  5.13 ± 0.72 4.48 ± 0.88 Note: *means significant level of 0.05,**means significant level of 0.01 compared with the control group

Results for the serum biochemistry of male rats are shown above in Table3. Albumin in the 4% male group shows value of 33.01±1.34 g/L, which wassignificantly lower than that of control group (p<0.05). The low contentof albumin in serum maybe due to synthesis deficiency. Two reasons maybe responsible for this change. Due to hepatitis, albumin absorption byliver may be decreased. The other reason may be the renal excretiondysfunction caused by low nephrogenic which may cause the excretion of alarge amount of albumin along with urine. Globulin in 4% male is28.1±2.77 g/L, which was significantly lower than that of control group(p<0.05). However, the value is in the normal range (15-28 g/L).Aspartate aminotransferase in 4% male was 91.17±8.13 U/L, which wassignificantly lower than that of control group (p<0.05). However, thevalue was also in the normal range (39-111 U/L). Alanineaminotransferase in 4% male was 39±7.73 U/L, which was significantlylower than that of control (p<0.05). However, the value was in thenormal range (20-61 U/L). Aspartate aminotransferase and alanineaminotransferase are the indicators for liver function. Increase in thetwo parameters may indicate some pathological change in liver anddecrease may be not clinically significant. Compared with control, allexperimental groups showed significantly lower amylase values (p<0.05),which was a benefit. Creatine kinase values in experimental groups weresignificantly lower than that of control groups (p<0.05), which was abenefit. Glucose in 4% male was significantly lower than that of controlgroup and was in the normal range (2.78-7.50 μmon). Calcium level in 1%and 4% male groups were significantly lower than that in control group(p<0.05). Decrease in calcium level may be due to (1) deficiency inparathyroid hormone; (2) Vitamin D deficiency or metabolic abnormality;(3) chronic kidney disease.

In conclusion, significant changes in serum biochemistry were mainlyobserved in the 4% male group and are mostly related with function ofliver or kidney.

Table 4 shows serum biochemistry for female rats in the 28-day toxicitystudy (n=10).

TABLE 4 0% 0.5% 1% 4% Albumin (g/L) 34.91 ± 1.28 34.53 ± 1.35 36.14 ±1.56 34.04 ± 1.11 Total protein (g/L) 64.32 ± 2.90 63.82 ± 3.95  68.02 ±4.31* 64.47 ± 3.50 Globulin (g/L) 29.42 ± 2.22 29.29 ± 3.29 31.88 ± 3.4730.42 ± 2.75 Globulin ratio  1.19 ± 0.09  1.19 ± 0.12  1.15 ± 0.13  1.13± 0.09 Total bilirubin (μmol/L) <1.0 <1.0 <1.0 <1.0 Aspartate  87.20 ±10.32 103.16 ± 8.70  99.50 ± 9.98 134.50 ± 5.44  Aminotransferase (U/L)Alanine 40.00 ± 4.50 41.62 ± 3.54 38.25 ± 3.86 40.28 ± 3.35Aminotransferase (U/L) Amylase (U/L) 1442.25 ± 148.93 1464.66 ± 147.65 1220.55 ± 125.14*  1191.37 ± 120.09* Creatinine (μmol/L) <4 <4 <4 <4Creatine kinase (U/L) 1054.60 ± 197.79 411.20 ± 43.84 540.50 ± 90.78363.00 ± 61.57 Triglyceride (μmol/L)  0.42 ± 0.21  0.43 ± 0.27  0.38 ±0.17  0.35 ± 0.09 Glucose (μmol/L)  8.66 ± 0.82  8.16 ± 1.48   6.89 ±0.58**   6.40 ± 1.02** Calcium (μmol/L)  2.49 ± 0.09  2.53 ± 0.07  2.55± 0.08  2.51 ± 0.11 Inorganic  2.48 ± 0.31  2.98 ± 0.37*  2.89 ± 0.23* 3.02 ± 0.42* phosphorus (μmol/L) BUN (μmol/L)  5.07 ± 0.97  4.91 ± 1.19 5.68 ± 0.52  5.61 ± 1.74 Note: *means significant level is 0.05,**means significant level is 0.01 compared with the control group

Results for serum biochemistry of female rats are shown in Table 4.Total protein in 1% female group was 68.02±4.31 g/L, which wassignificantly higher than that of the control group (p<0.05). However,it was in the normal range (53-69 g/L). Increased total protein levelmay be due to chronic liver disease. Amylase and glucose in 1% and 4%females were significantly lower than that of control group (p<0.05).Inorganic phosphorus level in 1% and 4% female groups were significantlyhigher than control group. However, they were in the normal range(1.87-3.6 μmon).

Hematology

Table 5 shows hematology for male rats in the 28-day toxicity study(number of animals=10).

TABLE 5 0% 0.5% 1% 4% RBC (10¹²/L)  6.26 ± 1.07  6.589 ± 0.46  6.63 ±0.20  6.50 ± 0.42 MCV (fL) 63.74 ± 2.64  62.85 ± 2.45 61.72 ± 1.91 61.73± 1.21 RDW % 21.64 ± 0.22  21.72 ± 0.37 21.73 ± 0.42 21.57 ± 0.22 RDWa(fL) 39.80 ± 2.18  39.08 ± 1.96 38.58 ± 1.82 40.11 ± 2.19 HCT % 39.82 ±2.72  41.42 ± 3.36 40.93 ± 1.11 40.11 ± 2.20 PLT (10⁹/L) 900.50 ± 27.501280.50 ± 97.06 1192.20 ± 104.45 1162.16 ± 98.46* MPV (fL)  6.96 ± 0.27  6.38 ± 0.40*  6.55 ± 0.29  6.39 ± 0.18* WBC (10⁹/l)  4.20 ± 2.19  4.60± 1.12  3.42 ± 1.07  3.88 ± 1.50 HGB (g/dL) 13.78 ± 2.14  14.13 ± 1.3014.22 ± 0.37 14.09 ± 0.77 MCH (pg) 22.10 ± 1.11  21.48 ± 1.11 21.43 ±0.67 21.71 ± 0.47 MCHC (g/dL)  0.78 ± 0.35  0.65 ± 0.27  0.21 ± 0.09 0.29 ± 0.09 LYM (g/dL)  3.44 ± 1.76  4.05 ± 1.06  2.98 ± 0.93  3.39 ±1.32 GRAN (g/dL)  0.62 ± 0.43  0.43 ± 0.30  0.33 ± 0.15  0.38 ± 0.23MONO (g/dL)  0.14 ± 0.05   0.12 ± 0.041 0.1 ± 0   0.11 ± 0.03 LYM %83.24 ± 4.25  90.76 ± 1.96* 87.23 ± 1.48 87.62 ± 3.62 GRA % 83.24 ± 4.25 88.30 ± 6.28 87.23 ± 1.49 87.62 ± 3.62 MON %  1.98 ± 0.24  1.47 ± 0.58 1.70 ± 0.45  1.57 ± 0.41 Note: *means significant level is 0.05, **means significant level is 0.01 compared with the control group

There were no treatment-related adverse effects of PWPC-GSH powder onhematology parameters in male rats. However, some statisticallysignificant differences occurred between control and treatment groups.PLT, MPV in 4% groups were significantly different from those of thecontrol (p<0.05). MPV and LYM in 0.5% group were also significantlydifferent from those of the control (p<0.05).

Table 6 shows hematology for female rats in the 28-day toxicity study(number of animals=10).

TABLE 6 0% 0.5% 1% 4% RBC (10¹²/L)  6.25 ± 0.30  6.53 ± 0.35  6.76 ±0.56*  6.61 ± 0.28 MCV (fL) 58.75 ± 2.07 59.71 ± 1.45 59.74 ± 2.29 59.26± 1.70 RDW % 20.95 ± 0.45 20.90 ± 0.38 20.86 ± 0.23 21.10 ± 0.39 RDWa(fL) 35.08 ± 1.46 35.87 ± 1.14 35.89 ± 1.87 35.88 ± 1.40 HCT % 36.73 ±1.78 38.96 ± 1.60 40.30 ± 2.64 39.11 ± 1.29 PLT (10⁹/L) 1098.00 ± 122.181271.00 ± 130.77 1330.57 ± 122.96 1286.00 ± 165.89 MPV (fL)  6.75 ± 0.73 6.30 ± 0.35  6.45 ± 0.38  6.46 ± 0.48 WBC (10⁹/L)  3.81 ± 2.17  4.64 ±1.34  5.86 ± 1.76  4.01 ± 0.93 HGB (g/dL) 13.18 ± 0.56 13.97 ± 0.55 14.46 ± 0.88*  14.10 ± 0.36* MCH (pg) 21.13 ± 0.61 21.41 ± 0.48 21.48 ±0.73 21.40 ± 0.63 MCHC (g/dL) 35.99 ± 0.43 35.87 ± 0.38 35.95 ± 0.4736.10 ± 0.35 LYM (g/dL)  3.19 ± 1.65  3.94 ± 1.13    5 ± 1.26  3.43 ±0.59 GRAN (g/dL)  0.51 ± 0.54  0.57 ± 0.25  0.70 ± 0.53  0.48 ± 0.38MONO (g/dL)  0.13 ± 0.05  0.14 ± 0.07  0.16 ± 0.07  0.13 ± 0.051 LYM %84.91 ± 5.25 84.80 ± 5.03 86.20 ± 4.43 85.94 ± 5.29 GRA % 84.91 ± 5.2584.80 ± 5.03 86.20 ± 4.43 85.94 ± 5.29 MON % 84.91 ± 5.25 84.80 ± 5.0386.20 ± 4.43 85.94 ± 5.29 Note: *means significant level is 0.05, **means significant level is 0.01 compared with the control group

There were no treatment-related adverse effects of PWPC-GSH powder onhematology parameters in female rats. However, some statisticallysignificant differences occurred between control and treatment groups.Compared with control, RBC in the 1% group showed significantly highervalue of 6.76±0.56×1012/L (p<0.05). This change may be caused by lack ofwater and should not be considered as test-substance related. HGB in the1% female group (14.46±0.88 g/dL) and the 4% female group (14.10±0.36g/dL) were significantly higher than control (p<0.05). However, thevalues were in the normal range (13.2-16.4 g/dL) and should not beconsidered as adverse effects.

Relative Organ Weights

Results for relative organ weights are shown in Tables 7 and 8. Malerats with PWPC-GSH powder showed significantly lower final body weightthen the control group (p<0.05). Compared with control, there was nosignificant difference in relative organ weights for organs for all ratsfed with PWPC-GSH powder except for liver and kidney in the 4% malegroup.

Table 7 shows relative organ weights for male rats in the 28-daytoxicity study (number of animals=10).

TABLE 7 0% 0.5% 1% 4% Body weight 296.8 ± 11.47  272.78 ± 10.28**    261± 12.59**   275 ± 18.70* Brain 5.35 ± 0.85 5.90 ± 2.11  6.45 ± 0.63*6.67 ± 0.67 Thymus 2.58 ± 0.60 3.01 ± 0.42 2.37 ± 0.38 2.66 ± 0.47 Heart3.80 ± 0.30 4.03 ± 0.50 3.92 ± 0.28 3.89 ± 0.28 Lung 4.74 ± 0.38 5.31 ±1.08 4.98 ± 0.52 4.78 ± 0.31 Liver 39.00 ± 4.33  37.22 ± 4.83  36.62 ±2.96  34.92 ± 2.83* Spleen 2.79 ± 0.92 2.78 ± 0.72 2.47 ± 0.35 2.79 ±0.39 Kidney 9.16 ± 0.70 9.32 ± 0.39 9.38 ± 0.38  9.66 ± 0.51* Bladder0.28 ± 0.04 0.31 ± 0.07 0.32 ± 0.05 0.29 ± 0.07 Testes 6.796 ± 1.79 8.62 ± 1.05 8.25 ± 0.23 8.67 ± 0.76 Epididymis 0.57 ± 0.11 0.48 ± 0.150.58 ± 0.09 0.59 ± 0.05 Seminal 1.95 ± 0.58 1.47 ± 0.42 1.11 ± 0.68 1.59± 0.50 Vesicle Note: *means significant level is 0.05, **meanssignificant level is 0.01 compared with the control group

For female rat, rats in the 4% group showed significantly lower finalbody weight (p<0.05). There was no significant difference in relativeorgan weights between rats fed with PWPC-GSH powder and control group.

Table 8 shows relative organ weights for female rats in the 28-daytoxicity study (number of animals=10).

TABLE 8 0% 0.5% 1% 4% Body weight 203.80 ± 10.14  205.20 ± 16.01  199.70± 11.93  192.10 ± 8.77*  Brain 7.14 ± 1.40 8.52 ± 0.86 8.22 ± 1.02 8.65± 0.84 Thymus 3.36 ± 0.46 3.31 ± 0.38 3.40 ± 0.52 3.31 ± 0.44 Heart 6.82± 0.86 3.99 ± 0.26 4.26 ± 0.37 4.08 ± 0.31 Lung 5.44 ± 0.55 5.09 ± 0.395.19 ± 0.35 5.13 ± 0.33 Liver 35.76 ± 2.74  38.28 ± 7.27  34.54 ± 2.14 36.08 ± 1.55  Spleen 2.51 ± 0.32 2.56 ± 0.40 2.32 ± 0.34 2.57 ± 0.21Kidney 8.83 ± 0.46 8.54 ± 1.53 8.70 ± 0.31 9.47 ± 0.51 Bladder 0.35 ±0.03 0.36 ± 0.07 0.35 ± 0.04 0.35 ± 0.06 Ovary 0.71 ± 023  1.65 ± 0.280.54 ± 0.16 0.56 ± 0.14 Uterus 2.11 ± 0.62 1.98 ± 0.62 1.75 ± 0.65 1.99± 0.98 Note: *means significant level is 0.05, ** means significantlevel is 0.01 compared with the control group

Pathology and Histopathology

FIGS. 10A and 10B show stained sections of several tissues in male andfemale rats in the 4% feeding group vs. control. Tissue samples wereprepared and stained by the usual method, and viewed by standardmicroscopic methods.

Conclusions

The results of PK studies indicated that the serum uptake of polymerizedwhey protein encapsulated GSH (PWP-GSH) was three times higher than thatof Kyowa's Setria GSH. The levels of GSH in brain and liver tissues forthe rats fed with polymerized whey protein encapsulated GSH comparedwith the group fed with commercial GSH diet were significant higherafter 3-4 hours of administration. Compared with control, there are somechanges in parameters of body weight, serum biochemistry and relativeorgan weights in the 4% feeding male group. Thus, it can be concludedthat the no-observed-adverse-effects level (NOAEL) was estimated to beat least 1% for male rats and 4% for female rats which are correspondingto about 50 and 200 folds of human daily intake value.

In summary, the bioavailability of whey protein encapsulated GSH(WP-GSH, PWP-GSH) was much improved compared with the standard controland it is a safe form of delivery system.

Example 3

Preparation of Polymerized Whey Protein Encapsulated Glutathione(PWP-GSH)

Whey protein concentrate (5 Kg) was dissolved in distill water at theconcentration of 10% (w/v) and stored at 4° C. overnight. The wheyprotein concentrate solution was then heated at 80° C. for 15 minutes.After cooling to room temperature, the PWPC solution was then mixed withGSH powder (5 Kg) at weight ratios of PWPC:GSH=1:1. The mixtures werestirred for 20 min for the complete dissolution. After blending, themixture was then freeze-dried to provide a PWP-GSH powder product.

Reduced glutathione (GSH) assay kit (A006-2-1), total antioxidativecapacity measurement kit (ABTS method) (A015-2-1) is available fromNanjing Jiancheng Bioengineering Institute (Nanjing, Jiangsu China). GSHcontent may be assayed using several methods. The following procedureprovided a determination of GSH content without digestion.

PWPC based GSH powder (2 g) was dissolved in 20 mL PBS buffer and thensonicated for 20 min for the complete extraction. After sonication, 1 mLsupernatant was taken and diluted for 800 folds. Then, 1 mL dilutedsolution was mixed with 1 mL protein removing agent and then centrifugedat 3500 rpm for 10 min. The supernatant was then determined for GSHcontent using GSH assay kit.

Alternatively, GSH content was determined after trypsin digestion.

Releasing solution: trypsin (10 g) with enzyme activity of 1:250 wasdissolved in 1 L NaCl solution (0.5%, w/v) and then adjusted to pH 8using 0.1 M NaOH solution. PWPC based GSH powder (0.3 g) placed into 30mL releasing solution and incubated while shaking at 100 rpm at 37° C.for 6 h. The mixture was then centrifuged at 5,000×g for 20 min and thenthe supernatant was diluted for 100 folds. The diluted suspension wasthen mixed with protein removing solution at ratio of 1:1 (v/v) and thencentrifuged. The supernatant was then determined for GSH content usingGSH assay kit.

Referring to FIG. 11 standard curve, without digestion, the OD values oftwo samples were 0.2883 and 0.2862, respectively, which correspond tothe content of 43.2% and 42.9% (w/w) of GSH in the PWPC-GSH product.After trypsin digestion, the OD values of the two samples were 0.2654and 0.2627, respectively, which correspond to 49.8% and 49.2% (w/w) ofGSH in the PWPC-GSH product.

Therefore, the processing technology for whey protein encapsulated GSH(PWP-GSH) manufacturing has been established and the recovery rate ofglutathione in the matrix was 99.2%, indicating that the loss of thisheat sensitive compound was only 0.8% throughout the process.

Example 4

Chemical characterization of PWP-GSH samples. It is well understood thatsamples made in accordance with the principles of this disclosure may becharacterized by various means well known in the art, including, but notlimited to, viscosity measurements and other rheological measurements,FT-IR, TEM/SEM photomicrography, microstructure and morphology studies,stability studies (solid phase, solution phase, humidity, thermal),particle size, Zeta potential, and the like. It is expected that thesaid chemical analyses will further show the unique qualities andproperties of the compositions described herein. Cf. Khan, et al., 2019.

Example 4A

1. Preparation of PWPC-GSH Using Whey Protein Concentrate (PWC)

PWPC-GSH system was prepared with advantages of simplicity, mildness,and organic solvent-free in comparison with other carriers based on polyisobutylcyanoacrylate, Eudragit RS 100/cyclodextrin and montmorillonite.In one characterization test measured in accordance with Zhang et al.(2021), PWPC exhibited bimodal pattern with two peaks at 594 nm and 4580nm with a wide particle size distribution (span of 9.22), consistentwith previous research. Combination with GSH (287.83±6.18 nm) slightlyincreased particle size (D50) from 1085±35.35 to 1115±7.07 nm withdecreased span from 9.22±0.22 to 6.86±0.19. Zeta potential for PWPC-GSHwas found to be 30.37±0.75 mV. The high surface charge endowed thePWPC-GSH system high stability since strong electrostatic repulsionbetween molecules prevents polymerization, precipitation, andflocculation. In addition, the positive surface charge of PWPC-GSH wouldfavor absorption in vivo since cell membranes carries negative charges.PWPC-GSH system displayed shear-thinning behavior in range of 1-300 s-1,indicating that interaction between droplets was weakened at highershear rate.

A DSC thermogram of GSH demonstrated an exothermic peak at 198° C. anddisappearance of this melting peak in the PWPC-GSH system, implying thatGSH was molecularly dispersed in PWPC particles. FTIR spectra analysisshowed that PWPC displayed an amide I (C═O vibration) spectrum peak at1654.39 cm-1 and red shift occurred after binding with GSH, indicatingthat PWPC was structurally changed and intermolecular hydrogen bondsformed. This PWPC-GSH system exhibited morphology of vermicularaggregates with its majority at a size of roughly 200 nm, with somelarger aggregates measuring upwards of approximately 400 nm, determinedby standard TEM imaging techniques (FIG. 12 ).

2. In Vivo Pharmacokinetic and Antioxidant Activity of PWPC-GSH

Whey protein has been widely studied as an effective means of nutrientdelivery due to its resistance to digestion by pepsin, its non-toxicnature, widely available sources and broad biocompatibility.Pharmacokinetic studies of the PWPC-GSH delivery system and free GSHwere conducted and plasma GSH concentration-time profiles for all groupswere determined. GSH concentration was observed to be the highest in theplasma of PWPC-GSH group, followed by free GSH, PWPC, and the controlgroup. The higher value in the plasma of mice gavage with PWPC-GSH thanthat of free GSH group may be due to the protection effect of highlyviscous PWPC by embedding GSH inside and preventing damage togastrointestinal enzymes and an acidic environment. These results wereconsistent with previous studies that the bioavailability of quercetinand vitamin D were improved through whey protein encapsulating.

Pharmacokinetic parameters were calculated using a mouse model. Comparedwith free GSH (maximum concentration (C_(max)) of 7.37 mg/L and areaunder the concentration-time curve (AUC) of 19.23 h×mg/L, higher C_(max)(19.41 mg/L) and AUC (48.63 h×mg/L) values were observed, indicating ahigher rate and degree of GSH absorption in the blood circulation inmice after administration with PWP-GSH. The 2.5-fold and 2.6-fold higherC_(max) and AUC in the PWPC-GSH group suggested that the PWPC-GSHdelivery system can improve the in vivo bioavailability of GSHeffectively vs. GSH in its pure form on its own. Whey protein alsoappears to possess a protective effect on GSH as a carrier duringabsorption into the intestinal tract which may be due to the resistanceto digestion by pepsin. In addition to delivery of the GSH itself, thewhey protein supplementation may have also contributed to the increasein GSH levels in vivo by virtue of the abundance of cysteine residueinherent to whey protein, which has the capability to pro-motebiosynthesis of GSH as a rate-limiting amino acid. The lower time tomaximum concentration (Tmax) (1 h) occurred in the PWPC-GSH group incomparison with that in free GSH (2 h), indicating less time wasrequired to reach the maximum concentration after administration. Theplasma concentration of GSH in the GSH group reached its maximum levelsafter 1.5 to 2 h which echoed data reported in the early literaturerelative to orally administered free GSH.

Total antioxidative capacity of samples at different time points wasmeasured using an assay kit. Antioxidant capacity of plasma in micegavage with PWPC-GSH was significantly higher than that of free GSHthrough the whole period (p<0.05). The first reason for the increasedantioxidant capacity of plasma after gavage of PWPC-GSH in mice was thatGSH concentration in plasma was improved using PWPC as a deliverycarrier. The second reason may be due to the antioxidant properties ofwhey protein. As measured by T-AOC (mM), the plasma antioxidant capacityof mice after gavage with PWPC was also slightly improved to a degreethat may or may not be consistent with an additive effect.

Example 4B

In accordance with Examples 3, 4 and 4A, various parameters weremeasured for PWPC-GSH and PWPI-GSC, compared with whey proteinconcentrate (WPC) and whey protein isolate (WPI) standards.

FIGS. 13A and 13B show particle size distribution of whey proteinencapsulated glutathione nanoparticles for WPC and WPI startingmaterials, respectively.

FIG. 14 shows polydispersity index (PDI) of whey protein encapsulatedglutathione nanoparticles for both WPC and WPI starting materials.

FIG. 15 shows measured Zeta potential (mV) of whey protein encapsulatedglutathione nanoparticles for both WPC and WPI starting materials.

FIGS. 16A and 16B show apparent viscosity and shear rate relationshipsof whey protein encapsulated glutathione nanoparticles for WPC and WPIstarting materials, respectively.

FIGS. 17A and 17B show circular dichroism of whey protein encapsulatedglutathione nanoparticles for WPC and WPI starting materials,respectively.

FIGS. 18A and 18B show FT-IR spectra of whey protein encapsulatedglutathione nanoparticles for WPC and WPI starting materials,respectively.

Example 5

Preparation of PWP-DIM. The procedure of Khan, et al. (2019) wasmodified as follows.

Components are added to a continuous scratch type stirred, temperature-and pH-controlled tank, or equivalent continuous stirred-type reactor(CSTR) system. Specifically, the tank is jacketed and connecting to asteam source. The temperature is controlled by a thermocouple. The otherparts include a pH probe on the side and thermometer at the bottom,along with a mechanical stirring system.

The whey protein was dispersed and polymerized in the tank before DIMwas added.

The temperature is ranged from 70 to 95° C., and pH range is from6.5-9.0.

The measured values of viscosity for the mix of the materials beforepolymerization and after are 100-300 mPas to 3000 mPas, respectively.

Results and Discussion.

DIM and whey protein concentrate are opposite in terms ofhydrophobicity/hydration properties. This process was designed toencapsulate the dispersed phase (DIM) with the polymerized whey proteinpolymers in the continuous phase. Upon stirring and heating, when theviscosity of the system reaches the desired range, the dispersed phasewas suspended and wrapped up by the polymers. The two phases of thematerials were formed as a continuous and uniformed micro gel oraggregates.

In the end, the reaction product was spray dried using the standardmethod and collected as an encapsulated powder.

Example 6

Chemical characterization of PWP-DIM samples. It is well understood thatsamples made in accordance with the principles of this disclosure may becharacterized by various means well known in the art, including, but notlimited to, viscosity measurements and other rheological measurements,FT-IR, TEM/SEM photomicrography, microstructure and morphology studies,stability studies (solid phase, solution phase, humidity, thermal),particle size, Zeta potential, and the like. It is expected that thesaid chemical analyses will further show the unique qualities andproperties of the compositions described herein. Cf. Khan, et al., 2019.

Example 7

In a similar manner, polymerized whey protein encapsulated coenzyme-Q10(PWP-CoQ10) was prepared by the above method using whey protein isolate(WPI) and characterized as an orange flaky powder. Assay (HPLC): 20.69%by weight.

As shown in FIGS. 19, 20, and 21 , the PWP-CoQ10 having various ratiosranging from 20:1 (PWPI:CoQ10) to 100:1 1 (PWPI:CoQ10) was characterizedby particle size (nm), polydispersity index (PDI), and Zeta potential(mV), respectively.

The use of the terms “a,” “an,” “the,” and similar referents in thecontext of describing the presently claimed invention (especially in thecontext of the claims) are to be construed to cover both the singularand the plural, unless otherwise indicated herein or clearlycontradicted by context. Recitation of ranges of values herein aremerely intended to serve as a shorthand method of referring individuallyto each separate value falling within the range, unless otherwiseindicated herein, and each separate value is incorporated into thespecification as if it were individually recited herein. Use of the term“about” is intended to describe values either above or below the statedvalue in a range of approximately ±10%; in other embodiments the valuesmay range in value either above or below the stated value in a range ofapproximately ±5%; in other embodiments the values may range in valueeither above or below the stated value in a range of approximately ±2%;in other embodiments the values may range in value either above or belowthe stated value in a range of approximately ±1%. The preceding rangesare intended to be made clear by context, and no further limitation isimplied. All methods described herein can be performed in any suitableorder unless otherwise indicated herein or otherwise clearlycontradicted by context. The use of any and all examples, or exemplarylanguage (e.g., “such as”) provided herein, is intended merely to betterilluminate the invention and does not pose a limitation on the scope ofthe invention unless otherwise claimed. No language in the specificationshould be construed as indicating any non-claimed element as essentialto the practice of the invention.

While in the foregoing specification this invention has been describedin relation to certain embodiments thereof, and many details have beenput forth for the purpose of illustration, it will be apparent to thoseskilled in the art that the invention is susceptible to additionalembodiments and that certain of the details described herein can bevaried considerably without departing from the basic principles of theinvention.

All references cited herein are incorporated by reference in theirentireties. The present invention may be embodied in other specificforms without departing from the spirit or essential attributes thereofand, accordingly, reference should be made to the appended claims,rather than to the foregoing specification, as indicating the scope ofthe invention.

We claim:
 1. A process for making a polymerized whey protein concentratemicroencapsulated glutathione, the process comprising the steps of: (a)dissolving a powder of whey protein concentrate (WPC) in deionized waterat room temperature to provide a WPC aqueous solution, wherein the WPCis present at concentration of 10 w/v %; (b) adjusting pH of the WPCaqueous solution to 7-8 and heating said WPC aqueous solution to about70-80° C. for about 15 minutes; (c) cooling the heated WPC aqueoussolution to room temperature to provide a polymerized WPC solution; (d)adding glutathione powder to the polymerized WPC solution to provide amixture; (e) stirring the mixture to achieve complete dissolution and toprovide a clear homogeneous solution; and (f) isolating the polymerizedWPC microencapsulated glutathione by freeze-drying or spray-drying toprovide a powder.
 2. The process of claim 1, wherein the and glutathioneare used in equal amounts by weight.
 3. The process of claim 1, whereina weight ratio of the WPC to glutathione is from about 1:1 to 1:2. 4.The process of claim 1, wherein the isolating step is spray-drying. 5.The process of claim 1, wherein the isolating step is freeze-drying.